Characterization of liver and cerebellar binding sites for avian pancreatic polypeptide.
Microsomal membranes from chicken liver and cerebellum specifically bind 125I-labeled avian pancreatic polypeptide (APP) with widely different affinities. To understand further the structural basis for this affinity difference as well as to determine the nature of the PP receptor, certain biochemical characteristics of chicken cerebellar and liver membrane [125I] APP-binding sites were determined. Trypsin digestion markedly reduced liver and cerebellar membrane binding of [125I]APP. Neuraminidase did not alter binding, while phospholipase-C lowered liver specific [125I]APP binding via a nonspecific digestion of the membrane. Cerebellar [25I]APP binding was unaltered by phospholipase-C. Dithiothreitol significantly inhibited liver and cerebellar specific [125I]APP binding without altering affinity. N-Ethylmaleimide (NEM) potently inhibited specific cerebellar [125I]APP binding and affinity and increased liver [125I]APP binding without altering affinity. NEM inhibited [125I]APP degradation by both liver and cerebellar membranes. NEM caused significant dissociation of [125I]APP from cerebellar membranes. Collectively, these studies indicate that chicken liver and cerebellar membrane [125I]APP-binding sites (either the putative receptors per se or the surrounding membranes) are proteinaceous and possess disulfide bonds important in ligand binding. Free thiol groups appear essential for cerebellar [125I]APP binding, while in liver membranes, free thiol groups interfere with binding or play no role in the binding process per se. These studies provide a foundation for a more precise molecular definition of the structures of PP receptors.
Adamo
M L
ML
Department of Biology, University of Houston, Texas 77204-5513.
Hazelwood
R L
RL
D000818:Animals; D001665:Binding Sites; D001717:Birds; D002531:Cerebellum / Q000201:enzymology / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D006868:Hydrolysis; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D010191:Pancreatic Polypeptide / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D010191:Pancreatic Polypeptide; D005033:Ethylmaleimide; D004229:Dithiothreitol